Shaped bone fiber-based products and a method of manufacture thereof

ABSTRACT

The present invention relates to shaped, bone fiber-based products and methods to make the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/639,902, filed on Mar. 5, 2015, which claims priority under 35 U.S.C.§ 119(e) to U.S. Provisional Patent Application Ser. No. 61/948,442filed Mar. 5, 2014, and U.S. Provisional Patent Application Ser. No.62/098,873 filed on Dec. 31, 2014. Each of these references areincorporated herein in their entirety by reference.

FIELD OF THE INVENTION

The present embodiments generally relate to shaped bone fiber-basedproducts and a method of manufacturing thereof.

DESCRIPTION OF THE PRIOR ART

Methods for manufacturing shaped, bone fiber-based products are known inthe prior art. The majority of these methods require the use ofexcipients, carriers and/or specialized drying conditions (pressure,lyophilization, etc.) to generate products of the desired shape andphysical properties. A need remains for a facile method of generatingshaped, fiber-based products with adequate physical properties of moldrelease, flexibility, compressibility, cohesion, and pliabilitypost-shaping.

U.S. Pat. No. 5,314,476 to Prewett et al. (incorporated in its entiretyby reference) discloses demineralized bone particles with a medianlength:median thickness of 10:1 incorporated into an osteogeniccomposition. The osteogenic composition maintains its cohesiveness andresists erosion subsequent to being applied to an osseous defect. Thecomposition contains between about 20-80 wt. % of a carrier (e.g.,glycerol) and describes producing particles of 10 mm length by 0.5 mmthick which are demineralized, and mixed with particles with glycerol toa putty-like consistency.

U.S. Pat. No. 5,507,813 to Dowd et al. (incorporated in its entirety byreference) discloses the production of sheets from elongate boneparticles, especially demineralized bone particles. The sheets compriseat least 60 wt. % bone particles of lengths of 10-100 mm, thicknesses of0.02-1 mm, and widths of 2-5 mm. Dowd describes a method of productionof the sheet using demineralized particles by spreading wet particles ona screen, applying a force (8 psi) while drying in an oven, and thencutting the sheet to size.

U.S. Pat. No. 6,436,138 to Dowd et al. (Dowd II) discloses a process forfabricating shaped material from demineralized bone particles similar tothe process disclosed in Dowd. However, the oven-drying step is alteredto finish with lyophilization prior to warming. The product may beformed in a desired shape or the sheet of particles may be positioned ona mold support.

U.S. Pat. No. 6,808,585 to Boyce et al. discloses an osteogenicosteoimplant in the form of a flexible sheet, which has a void volumethat is not greater than about 32%. The method to make the productcomprises providing a coherent mass of bone-derived particles, thenmechanically shaping the mass of particles to form a flexible sheet. Thebone particles are demineralized, lyophilized, and then mixed with abiocompatible carrier (50% by weight glycerol/water), then the mass iscompressively contacted by hand (rolled out) into a sheet of minimalthickness. The products of Boyce compared to Dowd supposedly had morevoid volume, less % demineralized bone matrix (DBM), greater elasticity(less ability to maintain shape).

U.S. Pat. No. 8,133,421 to Boyce et al. (Boyce II) discloses a method ofmaking an implant comprised of an aggregate of bone particles andoptional reinforces, where at least some of the bone particles are notfully demineralized. The aggregate is shaped into a coherent mass with abulk density of greater than approximately 0.7 g/cm³.

U.S. Pat. No. 7,582,309 to Rosenberg et al. discloses a DBM compositionof fibers with lengths between 250 μm to 2 mm and a biocompatible liquidto form a coherent, formable mass. The fibers are present in an amountgreater than 40 wt % of dry component.

U.S. Pat. No. 7,323,193 to Morris et al discloses a process todemineralize whole bone and thereafter subdividing the bone intoparticles by applying pressure to the bone with a mechanical press. Thepressure is applied from about 1000 to 40000 psi.

U.S. Patent Publication No. 2006/0030948 to Manrique et al. discloses anosteoimplant where demineralized bone particles are mechanicallyentangled with each other and are then shaped in a mold.

U.S. Patent Publication No. 2013/0136777 to Behnam et al. disclosesosteoinductive compositions comprising partially demineralized bonewhere the collagen structure is disrupted.

The present invention discloses products and methods that areadvantageous over this art as discussed below.

SUMMARY OF THE INVENTION

The disclosed invention is directed to a shaped fiber-based product anda method of manufacturing thereof. The shaped fiber-based productconsists of a three-dimensional form of dimensions established by theuse of a mold. The properties of the shaped fiber-based product provideimproved self-adhesion, flexibility and compressibility over relatedproducts in the prior art. The method of manufacturing relies onjudicious selection of fiber shapes and sizes, hydration with awater-miscible, low boiling solvent, molding, and drying of the shaped,fiber-based product. In some embodiments, the water-miscible, lowboiling solvent may be used in combination with water and/or awater-based solution. Fibers for the product may be cut from bone,foodstuffs, wood, plant-based materials, elastomers, thermoplastics, butare preferably cut from bone. In some embodiments, the fibers may beexposed to drying or lyophilization conditions. In some embodiments,demineralized bone fibers are shaped and sized to specific dimensions toenhance entanglement and subsequent final product self-adhesion,flexibility, and compressibility.

Another aspect of the invention is the use of a controlleddrying/lyophilization cycle following fiber molding to provide a productwith enhanced cohesiveness and flexibility. A further aspect of themethod is the use of a water-miscible, low boiling solvent prior thefinal drying of the fiber-based articles to provide improved fiberentanglement, fiber shaping, final product shape retention, and moldrelease of the final product. The use of a water-miscible, low boilingsolvent further enhances the desired final product properties ofself-adhesion, flexibility, and compressibility.

An aspect of the invention is a shaped, bone fiber-based product ofspecific dimensions. The product includes a plurality of bone fibers,where the average length of the fibers is between about 1 to about 200mm, and where the product is within between about 60% to about 100% ofat least one of a pre-dehydrated property upon rehydration. Thepre-dehydrated property can be shape, flexibility, or compressibility.

The product may be a cube, a block, a strip, or a sphere. The residualmoisture content of the product may be less than about 6%. Therehydrated product may be compressible to about 80% of an original sizeof the rehydrated product before dehydration. In some embodiments,following compression the rehydrated product substantially returns itsoriginal shape of the product before dehydration. In some embodiments,the rehydrated product remains greater than about 90% intact followingrehydration compared to its intact percentage before dehydration. Therehydrated product may remain greater than about 90% intact after it isbent, compressed, twisted, squeezed or rolled. The rehydrated productmay be bendable to about 90°. The rehydrated product may beosteoinductive. The rehydrated product may maintain at least oneproperty, where the property is shape, cohesiveness, pliability, orcompressibility, for at least one year after rehydration. The void tofiber ratio of the product may be between about 1:99 to about 1:11.

The fibers may be cortical bone, cancellous bone, or combinationsthereof. In some embodiments, the fibers may be fully demineralized,partially demineralized, mineralized or any combinations thereof. Thefibers may be partially dehydrated, fully dehydrated, or fully hydrated.The fibers may be allogeneic, autogeneic, and xenogeneic tissues, andcombinations thereof. The fibers may have a diameter of between about0.1 mm to about 30 mm. The fibers may be cut from a bone in a directionabout 15° to about 90° from the grain of the native collagen fibers.

The product may rehydrate in at least one aqueous liquid, and mayrehydrate within about 15 seconds. The aqueous liquid may be water,saline, buffer, balanced salt solution, blood, bone marrow aspirate,plasma, or combinations thereof.

An aspect of the invention is a method for forming a shaped, bonefiber-based product of specific dimensions. The method includes cuttingbone into fibers, entangling the bone fibers in an aqueous solution,placing the entangled fibers into a mold and drying the fibers in themold while warming under reduced pressure.

The product may be a cube, a block, a strip, or a sphere. The residualmoisture content of the product may be less than about 6%. Therehydrated product may be compressible to about 80% of an original sizeof the rehydrated product before dehydration. In some embodiments,following compression the rehydrated product substantially returns itsoriginal shape of the product before dehydration. In some embodiments,the rehydrated product remains greater than about 90% intact followingrehydration compared to its intact percentage before dehydration. Therehydrated product may remain greater than about 90% intact after it isbent, compressed, twisted, squeezed or rolled. The rehydrated productmay be bendable to about 90°. The rehydrated product may beosteoinductive. The rehydrated product may maintain at least oneproperty, where the property is shape, cohesiveness, pliability, orcompressibility, for at least one year after rehydration. The void tofiber ratio of the product may be between about 1:99 to about 1:11.

The fibers may be cortical bone, cancellous bone, or combinationsthereof. In some embodiments, the fibers may be fully demineralized,partially demineralized, mineralized or any combinations thereof. Thefibers may be partially dehydrated, fully dehydrated, or fully hydrated.The fibers may be allogeneic, autogeneic, and xenogeneic tissues, andcombinations thereof. The fibers may have a diameter of between about0.1 mm to about 30 mm. The fibers may be cut from a bone in a directionabout 15° to about 90° from the grain of the native collagen fibers.

The mold may be made from ceramic, aluminum, stainless steel, othermetals, and combinations thereof. The mold may have at least one openingthat may be a gap, a perforation, a screen, a slit, a shape andcombinations of openings. The mold may be capable of withstanding steamsterilization. The mold may have variable dimensions, which may bedetermined by an assessment of the void to be filled in the patient. Themold may include a lid, which may be attached to the mold or detachedfrom the mold.

The drying or dehydration step may be lyophilization. In someembodiments, the drying or dehydration step may include heating the moldto a temperature between about 30° C. to about 80° C. The dehydration ordrying step may include heating the mold under reduced pressure ofbetween about 1 nTorr to about 740 Torr. In some embodiments, the dryingor dehydration step may take place with airflow through the mold.

An aspect of the invention is a three-dimensional shape fiber-basedproduct of specific dimensions. The product includes a plurality offibers, with an average length between about 1 mm to about 200 mm. Thematerial for the fibers is bone, wood, food stuffs, plant-basedmaterials, elastomers, thermoplastics, or combinations thereof.

The product may be a cube, a block, a strip, or a sphere. The residualmoisture content of the product may be less than about 6%. Therehydrated product may be compressible to about 80% of an original sizeof the rehydrated product before dehydration. In some embodiments,following compression the rehydrated product substantially returns itsoriginal shape of the product before dehydration. In some embodiments,the rehydrated product remains greater than about 90% intact followingrehydration compared to its intact percentage before dehydration. Therehydrated product may remain greater than about 90% intact after it isbent, compressed, twisted, squeezed or rolled. The rehydrated productmay be bendable to about 90°. The rehydrated product may beosteoinductive. The rehydrated product may maintain at least oneproperty, where the property is shape, cohesiveness, pliability, orcompressibility, for at least one year after rehydration. The void tofiber ratio of the product may be between about 1:99 to about 1:11.

The fibers may be cortical bone, cancellous bone, or combinationsthereof. In some embodiments, the fibers may be fully demineralized,partially demineralized, mineralized or any combinations thereof. Thefibers may be partially dehydrated, fully dehydrated, or fully hydrated.The fibers may be allogeneic, autogeneic, and xenogeneic tissues, andcombinations thereof. The fibers may have a diameter of between about0.1 mm to about 30 mm. The fibers may be cut from a bone in a directionabout 15° to about 90° from the grain of the native collagen fibers.

The product may rehydrate in at least one aqueous liquid, and mayrehydrate within about 15 seconds. The aqueous liquid may be water,saline, buffer, balanced salt solution, blood, bone marrow aspirate,plasma, or combinations thereof.

An aspect of the invention is a method of forming a three-dimensionalshape fiber-based product of specific dimensions. The method includescontacting a plurality of fibers with a solvent. The solvent is watermiscible, low boiling solvent. The fibers are then placed into a moldand dehydrated to form the product.

The product may be a cube, a block, a strip, or a sphere. The residualmoisture content of the product may be less than about 6%. Therehydrated product may be compressible to about 80% of an original sizeof the rehydrated product before dehydration. In some embodiments,following compression the rehydrated product returns its original shapeof the product before dehydration. In some embodiments, the rehydratedproduct remains greater than about 90% intact following rehydrationcompared to its intact percentage before dehydration. The rehydratedproduct may remain greater than about 90% intact after it is bent,compressed, twisted, squeezed or rolled. The rehydrated product may bebendable to about 90°. The rehydrated product may be osteoinductive. Therehydrated product may maintain at least one property, where theproperty is shape, cohesiveness, pliability, or compressibility, for atleast one year after rehydration. The void to fiber ratio of the productmay be between about 1:99 to about 1:11.

The solvent mixture may include water and the water miscible, lowboiling solvent. In some embodiments, the solvent may include awater-based solution and the water miscible, low boiling solvent. Thewater-based solution may be an aqueous acid, an aqueous base, a balancedsalt solution, a buffer, or combinations thereof. In some embodiments,the ratio of the water-based solution is mixed with the water miscible,low boiling solvent may be between about 1:99 to about 99:1. The watermay be mixed with the water miscible, low boiling solvent in a ratio ofabout 1:99 to about 99:1. The water miscible, low boiling solvent may beacetone, acetonitrile, ethanol, methanol, tetrahydrofuran, orcombinations thereof. In some embodiments, the water miscible, lowboiling solvent may be ethanol. In some embodiments, the fibers may becontacted with ethanol and water. In some embodiments, the fibers may becontacted with the solvent prior to or after placement in the mold.

When the fibers contact the water miscible, low boiling solvent, thefiber curl, fiber twist, and/or fiber entanglement may increase.

The material for the fibers may be bone, wood, foodstuff, plant-basedmaterial, an elastomer, a thermoplastic, or a combination thereof. Ifthe material is bone, then the bone may be cortical bone, cancellousbone, or combinations thereof. The bone may be demineralized, partiallydehydrated, or completely hydrated.

The mold may be made from ceramic, aluminum, stainless steel, othermetals, and combinations thereof. The mold may have at least one openingthat may be a gap, a perforation, a screen, a slit, a shape andcombinations of openings. The mold may be capable of withstanding steamsterilization. The mold may have variable dimensions, which may bedetermined by an assessment of the void to be filled in the patient. Themold may include a lid, which may be attached to the mold or detachedfrom the mold.

The drying or dehydration step may be lyophilization. In someembodiments, the drying or dehydration step may include heating the moldto a temperature between about 30° C. to about 80° C. The dehydration ordrying step may include heating the mold under reduced pressure ofbetween about 1 nTorr to about 740 Torr. In some embodiments, the dryingor dehydration step may take place with airflow through the mold.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a perspective graphical view of a shaped fiber-basedproduct manufactured by the method of the present invention;

FIG. 2 illustrates a perspective graphical view of the method of form ashaped fiber-based product in accordance with the present invention;

FIG. 3 illustrates an ethanol-treated shaped fiber-based productproduced by an exemplary embodiment of the disclosed method and anuntreated product retained within the shaping molds; and

FIG. 4 illustrates an ethanol-treated shaped fiber-based productproduced by an exemplary embodiment of the disclosed method and anuntreated product;

FIG. 5A illustrates a rehydrated product produced by methods known inthe prior art; and

FIG. 5B illustrates a rehydrated product produced by an exemplaryembodiment of the disclosed method.

FIG. 6 is a chart, which represents levels of insulin-like growthfactor-1 (IGF-1) in three samples of demineralized cancellous bonematrix from the same donor after subjecting the three samples todifferent demineralization protocols. Samples A and B representdemineralized cancellous bone matrix produced according to theprocesses, methodologies, and techniques of the present invention.Sample C represents demineralized cancellous bone matrix producedaccording to the state of the prior art found in the literature,demonstrating damage or removal of IGF-1.

FIGS. 7a-7c illustrate the effect of acid exposure on the levels ofnative growth factors in demineralized cancellous bone. FIG. 7aillustrates the effect of acid exposure on BMP-2. FIG. 7b illustratesthe effect of acid exposure on BMP-4. FIG. 7c illustrates the effect ofacid exposure on BMP-7. Sample A represents the growth factor content ofdemineralized cancellous bone matrix processed according to a preferredembodiment of the present invention, showing higher levels of BMPsversus samples B and C. Sample B represents the growth factor content ofdemineralized cancellous bone matrix processed according to methodscurrently being practiced in the industry, showing reduced levels ofBMPs in comparison with sample A representing the current invention.Sample C represents the growth factor content in demineralizedcancellous bone matrix processed by exposure to acid for 24 hours,demonstrating damage or removal of BMPs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a shaped bone fiber-based product andmethods of making the same.

“Allogeneic” or “allograft”, as used herein, refers to tissue derivedfrom a non-identical donor of the same species, which may be a DBM.

“Autogeneic” or “autograft”, as used herein, refers to tissue derivedfrom and implanted into the same identical patient.

“Biocompatible”, as used herein, refers to the property of beingbiologically compatible with a living being by not causing harm.

“Osteoinductive”, as used herein, refers to the ability of a material toinduce bone healing via recruitment of osteoprogenitor cells.

“Patient”, as used herein, refers to a living recipient of thebiomaterial-based implants of the present invention.

“Xenogeneic” or “xenograft”, as used herein, is defined as tissuederived from a non-identical donor of a different species.

The shaped fiber-based products of the invention have many advantagesover the prior art. The rehydrated fiber-based products of the inventioncompress under a force of between about 10 g-force/square cm to about4000 g-force/square cm. The rehydrated, shaped fiber-based product maybe compressible to about 80% of its original size, to about 60% of itsoriginal size, to about 20% of its original size, to about 5% of itsoriginal size without loss of structural integrity or fiber cohesion.Upon removal of an external compressing force, the products return totheir original shape. The shaped fiber-based product may also rehydraterapidly within an aqueous fluid over a period of about 15 seconds toabout 30 minutes, of about 1 minute to about 25 minutes, or of about 5minutes to about 20 minutes. In some embodiments, the shaped fiber-basedproduct may also have a high rehydration rate of between about 0.5 mL ofliquid/g of product/minute to about 10 mL of liquid/g of product/minute.Suitable aqueous fluids include, but are not limited to, water, saline,buffer, balanced salt solution, blood, bone marrow aspirate, plasma andcombinations thereof.

The shaped product is not a putty, but rather a sponge-like material maybe composed of a single material or a mixture of materials which may beused as scaffolding during bone regrowth. In some embodiments, theshaped product may be composed of solely bone tissue. Furthermore, whilethe invention may be used to produce a sheet that may later be cut toform a specific shape, the invention allows for the shape to be formedwithout this additional step of cutting. The base material of theinvention is fibers rather than particles, for example bone fibersrather than bone particles. Furthermore, the fiber to void ratio may bedetermined and maintained because an external force is not requiredduring the formation of the shaped fiber base product of the presentinvention.

Utilizing the drying/lyophilization parameters of the invention, thefinal shape of the product is within about 10% of its projected sizebased on the mold. Finally, the void to fiber ratio may be controlled tobetween about 1:99 to about 1:1, with a preferred void to fiber ratioranging from about 1:19 to about 1:3. In some embodiments, the void tofiber ratio may be between about 1:99 to about 1:11, about 1:75 to about1:4, between about 1:25 to about 1:5, between about 1:20 to about 1:6,or between about 1:10 to about 1:5.

In some embodiments, the method for making the shaped fiber-basedproduct utilizes low boiling solvents to facilitate final drying of thefiber-based material via azeotropic drying. The method for making theshaped fiber-based product also advantageously releases the finalproduct from a mold with minimal if any damage or breakage. Furthermore,because of the advantageous mold release, the shape of the shapedfiber-based product is retained after removal. Thus, the final shape iswithin about 10% of its projected size based on the mold. Finally, thefiber to void ratio may be controlled to between about 1:99 to about1:1, with a preferred void to fiber ratio ranging from about 1:19 toabout 1:3. In some embodiments, the void to fiber ratio may be betweenabout 1:99 to about 1:11, about 1:75 to about 1:4, between about 1:25 toabout 1:5, between about 1:20 to about 1:6, or between about 1:10 toabout 1:5.

FIG. 1 illustrates a perspective graphical view of a shaped fiber-basedproduct 1 formed by the method of this invention. The shaped fiber-basedproduct may be shaped in the form of a block as shown, or in the form ofa cube, strip, sphere, or other three-dimensional shape as desired. Theshape of the product may be uniform or irregular as desired by theend-user of the article. In some embodiments, the size of the productmay be larger than the final desired product and cut to a desireddimension. As illustrated in FIG. 2, the fiber-based material of theproduct may be formed from fiber components in a regular 2 a orirregular arrangement 2 b. In some embodiments, the fibers may be placedin a consistent, parallel orientation 2 a. In some embodiments, thefibers of the product may be entangled and interlaced in multipleorientations 2 b to provide strength and adhesion of the fibers to oneanother within the resultant product. In some embodiments, the shapedproduct 1 may contain voids within the fiber-based material. The void tofiber ratios may vary from about 1:99 to about 1:1, with a preferredvoid to fiber ratio ranging from about 1:19 to about 1:3. In someembodiments, the void to fiber ratio may be between about 1:99 to about1:11, about 1:75 to about 1:4, between about 1:25 to about 1:5, betweenabout 1:20 to about 1:6, or between about 1:10 to about 1:5.

The fibers that comprise the shaped product 1 may consist of bone, wood,food stuffs, plant-based materials, elastomers, thermoplastics, orcombinations thereof. These fibers may be fully or partially solvated asneeded to form a product of the desired moisture level and physicalproperties. The fibers within the shaped product 1 may be of lengths ofabout 1 mm to about 200 mm, of about 2 mm to about 150 mm, of about 5 mmto about 70 mm, to about 10 mm to about 60 mm. The average length of thefibers may be between about 15 mm to about 50 mm, in some embodimentsabout 30 mm. The fibers may have a width or diameter of about 0.1 mm toabout 30 mm, of about 0.2 mm to about 15 mm, of about 0.5 mm to about 10mm, to about 1 mm to about 8 mm. The average width or diameter may bebetween about 1 mm to about 5 mm, in some embodiments between about 2-3mm. The thickness of the shaped product may be between about 0.01 mm toabout 1 mm. In some embodiments, the thickness may be about 0.01 mm,about 0.05 mm, about 0.1 mm, about 0.2 mm, about 0.25 mm, about 0.3 mm,about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm,about 0.9 mm, about 0.95 or about 1 mm. The shaped product 1 is composedof bone fibers, the bone may be cortical, cancellous, or a combinationof the two bone types. The bone may be allogeneic, autogeneic, orxenogeneic. In some embodiments, other materials may be entangled withinor added to the fibers (e.g., bone chips, biocompatible minerals, andBioGlass). The bone fibers for the invention may be generated by avariety of methods and techniques known in the prior art, for exampleU.S. Pat. No. 5,314,476, which is incorporated in its entirety byreference. In some embodiments, the bone is cut into fibers in an angleto the plane of the native collagen fibers within the bone, wherein thecutting blade is at an angle of about 30° to 90°, of about 45° to 90°,or of about 60° to 90°. The fibers comprising the shaped product 1 maybe mineralized, fully demineralized, partially demineralized, or acombination of the foregoing. Demineralized bone matrix for use by thedisclosed method may be prepared using any method or techniques known inthe art, for a typical demineralization protocol. Exampledemineralization protocols are disclosed in U.S. Pat. No. 5,314,476, or8,574,825, each reference is incorporated by reference in theirentirety. In some embodiments, the demineralization protocol for thebone fibers may be optimized based on the bone fiber type to provide themaximum level of growth factors, such as bone morphogenetic proteins(BMPs). FIG. 6 illustrates an IGF-1 concentration of greater than 60 pgof IGF per gram of demineralized bone matrix. FIG. 7a illustrates aBMP-2 concentration of greater than 2700 pg of BMP-2 per gram ofdemineralized bone matrix. FIG. 7b illustrates a BMP-4 concentration ofgreater than 200 pg of BMP-4 per gram of demineralized bone matrix. FIG.7c illustrates a BMP-2 concentration of greater than 9000 pg of BMP-7per gram of demineralized bone matrix. Following any necessarypre-treatment (e.g., demineralization), the fibers 2 may be contactedwith a water miscible, low boiling solvent. In some embodiments, thewater miscible, low boiling solvent may be used in combination withwater and/or water-based solutions (e.g., buffers). The contact methodfor the fibers 2 and the solvent may consist of pouring the solvent overthe fibers, soaking the fibers in the solvent, forming a slurry of thefibers in the solvent, or a combination of these contact methods. Insome embodiments, the ratio of solvent volume to mass of the fibers maybe selected from a range of about 0.5 mL of solvent/gram of fibers toabout 30 mL solvent/gram of fibers, from about 1 mL of solvent/gram offibers to about 20 mL solvent/gram of fibers. Judicious selection of thesolvent volume to mass of the fibers allows tuning of the void to fiberratio within the final fiber-based material. In some embodiments, thedemineralized bone fibers may have a residual calcium level of less thanabout 8%, less than about 6%, or less than about 2%. Suitable watermiscible, low boiling solvents include, but are not limited to, acetone,acetonitrile, ethanol, methanol, tetrahydrofuran, and combinationsthereof. In some embodiments, the water miscible, low boiling solventsfrom a low boiling azeotrope with water to facilitate final productdrying. Suitable water-based solutions included, but are not limited to,aqueous acids, aqueous bases, balanced salt solutions, and buffers.Suitable aqueous acids include, but are not limited to, acetic acid,ascorbic acid, hydrochloric acid, citric acid, sulfuric acid, formicacid, succinic acid, and lactic acid. The concentration of the aqueousacids may range from 0.01 to 10 M. Suitable aqueous bases include, butare not limited to, hydroxides, carbonates, phosphate, and ammonia. Theconcentration of the aqueous bases may range from about 0.01 to about10M. Counter ions of the aqueous bases include, but are not limited to,sodium, potassium, calcium, and ammonium. Suitable balanced saltsolutions and buffers include, Hank's balanced salt solution, phosphatebuffered saline, and saline.

A method of forming the shaped fiber-based products 1 may consist ofplacing the fibers 2, into contact with a water miscible, low boilingsolvent. By contacting the fibers with a water miscible, low boilingsolvent, the fibers may become further curled, twisted, shriveled, or acombination thereof. The increased curling, twisting, and/or shrivelingof the fibers by a water miscible, low boiling solvent promotes fiberentanglement and subsequent shape retention of the final product. Thefibers thus contacted with solvent are then placed within a mold 4 asshown in FIG. 2. Following any necessary pre-treatment (e.g.,demineralization), the fibers 2 may be placed within a mold 4 asillustrated in FIG. 2. The mold 4 is capable of forming athree-dimensional shape. In some embodiments, the mold may fully enclosethe fibers, or may have a lid 3 if desired. The lid 3 may be attached tothe mold, detachable, or separate from the mold. The mold 4 and lid 3may be perforated to fully or partially to allow removal of moisturefrom the fibers 2 during shaping. In other embodiments, the mold may beused to form a three-dimensional shape, for example a sheet of material,which may be further shaped. The mold 4 may be composed of various heatresistant materials such as, but not limited to, ceramics, elastomers,aluminum, stainless steel, thermoplastics, any other metals, orcombinations thereof. The mold 4 may be amenable to steam sterilization.The mold 4 dimensions may be pre-set or adjustable to the desired finalproduct dimensions. The mold 4 may be constructed of a screen-likematerial. The mold 4 may have a non-stick coating, such as Teflon. Themold 4 or mold lid 3 may apply adjustable inward pressure.

In some embodiments, the mold 4 may have drainage holes or openings toallow moisture to enter and exit the product during use. In someembodiments, the mold 4 may have openings or drainage holes at least onone side 5. In another embodiment, the mold may be comprised of onlythree sides so that moisture may exit from open sides of the mold 4. Inanother embodiment, the mold 4 may be composed of a screen with numerousopenings to allow moisture entry or exit during use. In otherembodiments, the mold 4 may be a sieve or strainer.

The shaped product 1 may be shaped specifically to fill a void. The voidmay be determined by pre-assessment of a void, such as a bone voidwithin a patient. The final use of the shaped product 1 may be placementwithin a void of the patient.

During the forming of the shaped product 1, the fibers may be laid in aregular pattern 2 a within the mold 4, or entangled as a mesh, braid, orinterwoven in some manner 2 b prior to placement within the mold 4. Insome embodiments, the fibers may be laid or entangled around anotherarticle. If the fibers are placed around another article, the containedarticle may be composed of the same material or of materials of adifferent composition than the surrounding fibers (e.g., a shapedfiber-based article composed of demineralized cancellous fibers may beplaced inside fibers composed of demineralized cortical bone). Thecontained article may be composed of elastomers, ceramics, metals, metalalloys, or plastics.

The fibers 2 are contacted with a solution containing a water miscible,low boiling solvent prior to placement or after placement in the mold 4.The solution containing a water miscible, low boiling solvent, thefilled mold may be held at temperatures ranging from about −10° C. toabout 70° C.; preferably, the solution will be used at room temperature.In some embodiments, the fibers may be contacted with a combination ofwater and a water miscible, low boiling solvent prior to placement orafter placement in the mold 4. The ratio of water to the water miscible,low boiling solvent may range from about 0:100 to about 99:1, from about5:95 to about 95:1, to about 50:50. In other embodiments, the fibers maybe contacted with a combination of a water-based solution and a watermiscible, low boiling solvent prior to placement or after placement inthe mold 4. The ratio of the water-based solution to the water miscible,low boiling solvent may range from about 0:100 to about 99:1, from about5:95 to about 95:1, to about 50:50. In a preferred embodiment, thefibers 2 may be slurried in a solution containing a water miscible, lowboiling solvent prior to placement within the mold 4. After the fibersare placed in the mold 4, a lid 3 may be placed on the mold 4 ifdesired. Excess moisture will be allowed to drain from the fibers withinthe mold 4.

The apparatus may be placed into a drying chamber 6 in a frozen orthawed state. The drying step may consist of blowing gas through themold and/or subjecting the apparatus to reduced pressure, heating,lyophilization (under reduced pressure), or a combination of heating andvacuum. The gas used may include, but is not limited to, nitrogen,helium, argon, and combinations thereof. The drying may be performedunder reduced pressure between about 1 nTorr and about 740 Torr. Thedrying step may consist of, or include, air flow into or through themold. In some embodiments, the mold 4 or mold lid 3 may provideuser-adjustable pressure to allow variance of the compaction of theresultant article. This user-adjustable pressure of the mold 4 or moldlid 3 may allow for articles of varied “sponginess” or flexibility.Drying may include heating the material to a temperature between about30° C. to about 80° C., in some embodiments about 40° C. Followingdrying, the mold may be removed from the drying chamber 6, and theshaped product 1 may be removed from the mold. In some embodiments, theuse of fibers 2 slurried in a solvent containing a water miscible, lowboiling solvent prior to placement within the mold 4 results in anarticle with improved shape retention and with improved mold releasecharacteristics.

In some embodiments, following forming of the bone fibers into the mold4, the filled mold may be frozen at a temperature of about −100° C. toabout 0° C., of about −90° C. to about −10° C. to about −80° C. to about−20° C. The apparatus may be placed into a drying chamber 6 in a frozenor thawed state. The drying step may include subjecting the apparatus toreduced pressure, heating, lyophilization (under reduced pressure), or acombination of dehydration and lyophilization. In preferred embodiments,the drying/lyophilization step is performed under reduced pressures ofabout 0.100 Torr to about 600 Torr, about 0.800 Torr to about 400 Torr,about 1.8 Torr to about 300 Torr, or any sub-range within the largestrange. Drying may include heating the material to a temperature betweenabout 30° C. to about 80° C., in some embodiments about 45° C. Thetemperature of the drying step may change over the time period of thedrying from about 80° C. to −80° C., from about 60° C. to −70° C., fromabout 50° C. to 0° C., from about 45° C. to 25° C. Drying may take placeover the range of about 1 hour to about 48 hours, of about 3 hours toabout 30 hours, of about 4 hours to about 25 hours. During the dryingstep, the vacuum may be increased from an initial pressure of about 100Torr to about 600 Torr, of about 200 Torr to about 400 Torr, of about300 Torr; to a pressure of about 100 mTorr to about 30,000 mTorr, ofabout 800 mTorr to about 3000 mTorr, of about 1800 mTorr, or any othersub-range within the larger range. Following drying, the mold may beremoved from the drying chamber 6, and the shaped product 1 may beremoved from the mold. In some embodiments, the drying of fibers 2within the mold 4 under the conditions described results in an articlewith improved shape retention and enhanced cohesiveness and flexibilityupon rehydration. In the preferred embodiments, the drying of the fibers2 within the mold 4 under the conditions described results in an articlewith retained osteoinductivity and a residual moisture content of lessthan about 6%, less than about 4%, or less than about 2%. In someembodiments, at least one rehydrated product property may be withinabout 60% to about 100% of its original property following rehydration.For example, the rehydrated product may be within about 75% of itsinitial shape, flexibility or compressibility prior to dehydration. Insome embodiments, the rehydrated product may be within about 60%, about65%, about 75%, about 85%, about 90%, about 95%, about 99% of itsinitial shape, the initial flexibility or the initial compressibility.

Table 1 illustrates several different drying protocols, each of whichare suitable with the invention. All values provided in Table 1 areapproximate.

TABLE 1 Drying protocols Protocol Step Protocol 1 Protocol 2 Protocol 3Step 1 45° C., 300 torr, 45° C., 300 torr, 300 min 45° C., 300 torr, 300min 300 min Step 2 35° C., 300 torr, 45° C. → 35° C., 300 torr, 45° C. →35° C., 300 torr, 900 min 900 min 720 min Step 3 25° C., 300 torr 25°C., 300 torr 35° C., 2450 mtorr, 180 min until stopped until stoppedStep 4 25° C., 2450 mtorr, 10 min Step 5 25° C., 2450 mtorr untilstopped

When bone fibers are demineralized and subjected to thelyophilization/drying conditions described in the existing art, thefinal shaped article fails to retain its shape, cohesiveness,pliability, and compressibility following rehydration within aqueousliquids of more than a few minutes or in some instances rehydrationtimes of more than a few seconds, as illustrated in FIG. 3A. When theshaped, bone fiber-based products of the present invention are subjectedto rehydration, the products retain their shape, cohesiveness,pliability, and compressibility. The products formed by the methods ofthe invention remain greater than about 90% intact upon rehydration inaqueous liquids. The products formed by the methods of the invention arebendable to greater than about 90° upon rehydration in aqueous liquids.The products formed by the methods of the invention display the highlydesired properties of shape retention, cohesiveness, pliability, andcompressibility upon rehydration for time periods of about 10 minutes toabout 1 year, of about 1 hour to about 6 months, of about 3 hours toabout 1 month. Suitable aqueous liquids for rehydration include, but arenot limited to, water, salines, buffers, balanced salt solutions, blood,and bone marrow aspirate.

EXAMPLE Example 1

A section of bovine cortical bone was shredded into fibers of an averagelength of about 15 mm and a average thickness of 1.0 mm using a boneshard cutter. The bone fibers were then demineralized following amodification version of the demineralization steps described in U.S.Pat. No. 5,314,476, which is incorporated in its entirety by reference.Briefly, the fibers were slurried in about 70 wt. % ethanol (about 30mL/g of bone) for about one hour at room temperature. The ethanol wasdecanted off the fibers. Then about 0.6 N HCl was added to the fibers(about 15 mL/g of bone). The acid mixture was stirred for three hours atabout room temperature. Following decanting of the acid, the fibers werecovered and rinsed three times with water. The water for each rinse wasreplaced at about five-minute intervals. Following decanting of thefinal water rinse, the fibers were covered with about 0.1 M sodiumphosphate and soaked at about room temperature until the pH of thesolution was greater than about 6.8. Following decanting of the sodiumphosphate solution, the fibers were rinsed two times with water. Thewater for each rinse was replaced at about five-minute intervals. Thefibers were then split into two groups for comparison.

One group of the demineralized fibers were slurried in water for aboutfive minutes at about room temperature and then placed directly in amold and the excess water was allowed to drain from the mold. The moldcontaining the “untreated” fibers was then placed into an oven and heldat about 40° C. for about eight hours.

The other group of demineralized fibers were slurried in 100% ethanolfor about five minutes at about room temperature. The ethanol wasdecanted from the fibers and the fibers were placed in a mold. Theexcess liquid was allowed to drain from the mold. The mold containingthe ethanol-treated fibers was then placed into an oven and held atabout 40° C. for about eight hours.

After eight hours, the molds were removed from the oven. FIG. 3illustrates the two samples within the molds. As illustrated in FIG. 3,the ethanol-treated product retained its shape during drying and beganto partially release from the mold, facilitating removal of the productfrom the mold. The untreated product collapsed during drying and adheredto the underlying mold. FIG. 4 shows the two shaped fiber-based productsreleased from the molds. As shown in FIG. 4, the ethanol-treated productretained the regular shape of the underlying mold more than theuntreated product.

Example 2

A section of cortical bone was shredded into fibers of an average lengthof about 15 mm and an average thickness of about 1.0 mm using a boneshard cutter. The mineralized bone fibers were then demineralizedfollowing a modified version of the demineralization steps described inU.S. Pat. No. 5,314,476, which is incorporated in its entirety byreference. Following acid demineralization and neutralization of theacid, the moisture-saturated fibers were poured into perforated metalmolds. The excess liquid was allowed to drain out of the molds

The fiber-containing molds were split into two groups for comparison.Each group of molds were subjected to two drying/lyophilizationconditions (see Table 2 below for drying/lyophilization conditions). Allof the values given in Table 2 are approximate.

TABLE 2 Drying/Lyophilization Conditions Group A Products Group BProducts Shelf Shelf Temperature Time Vacuum Temperature Time Vacuum (°C.) (minutes) Pressure (° C.) (minutes) Pressure −60 30 200 mT 45 300300 Torr 37 1080  50 mT 35 900 300 Torr

After treatment to the conditions detailed in Table 2, the shaped, bonefiber-based products were removed from the molds. Bothdrying/lyophilization conditions provided products that were shaped bythe molds. However, upon rehydration in saline for less than tenminutes, the bone fibers of the Group A products began to completelydissociate (see FIG. 5A). The Group B products remained >90% intact,pliable, and compressible for greater than one week in the fullyhydrated state (see FIG. 5B).

The foregoing description of the present invention has been presentedfor purposes of illustration and description. Furthermore, thedescription is not intended to limit the invention to the form disclosedherein. Consequently, variations and modifications commensurate with theabove teachings, and the skill or knowledge of the relevant art, arewithin the scope of the present invention. The embodiment describedhereinabove is further intended to explain the best mode known forpracticing the invention and to enable others skilled in the art toutilize the invention in such, or other, embodiments and with variousmodifications required by the particular applications or uses of thepresent invention. It is intended that the appended claims be construedto include alternative embodiments to the extent permitted by the priorart.

1. A shaped, bone fiber-based product of specific dimensions comprising:a plurality of bone fibers, wherein an average length of the pluralityof fibers is between about 1 and about 200 mm, and wherein the productis within between about 60% to about 100% of at least one of apre-dehydrated shape, a pre-dehydrated flexibility, and a pre-dehydratedcompressibility upon rehydration.
 2. The shaped, bone fiber-basedproduct of claim 1, wherein the shape is selected from the groupconsisting of a cube, a block, a strip, and a sphere.
 3. The shaped,bone fiber-based product of claim 1, wherein the plurality of bonefibers are selected from the group consisting of cortical bone,cancellous bone, and combinations thereof.
 4. The shaped, bonefiber-based product of claim 1, wherein the plurality of bone fibers areat least one of a fully demineralized bone, a partially demineralizedbone and a mineralized bone.
 5. The shaped, bone fiber-based product ofclaim 1, wherein the plurality of bone fibers are at least one of apartially dehydrated, a fully dehydrated, or a fully hydrated.
 6. Theshaped, bone fiber-based product of claim 1, wherein a void to fiberratio is between about 1:99 to about 1:11.
 7. The shaped, bonefiber-based product of claim 1, wherein the plurality of bone fibershave diameter of between about 0.1 mm to about 30 mm.
 8. The shaped,bone fiber-based product of claim 1, wherein the plurality of bonefibers are selected from the group comprising allogeneic, autogeneic,and xenogeneic tissues, and combinations thereof.
 9. The shaped, bonefiber-based product of claim 1, wherein the plurality of bone fibers arecut from a bone in a direction about 15° to about 90° from the grain ofthe native collagen fibers.
 10. The shaped, bone fiber-based product ofclaim 1, wherein a residual moisture content of the product is less thanabout 6%.
 11. The shaped, bone fiber-based product of claim 1, whereinthe rehydrated product is bendable to about 90°.
 12. The shaped, bonefiber-based product of claim 1, wherein the rehydrated product isosteoinductive.
 13. The shaped, bone fiber-based product of claim 1,wherein the void to fiber ratio is between about 1:19 and about 1:3. 14.The shaped, bone fiber-based product of claim 1, wherein the rehydratedproduct remains greater than about 90% intact following rehydrationcompared to its intact percentage before dehydration.
 15. The shaped,bone fiber-based product of claim 1, wherein the rehydrated productremains greater than about 90% intact after the rehydrated product isbent, compressed, twisted, squeezed or rolled.
 16. The shaped, bonefiber-based product of claim 1, wherein the rehydrated productcompresses under a force of between about 10 g-force/square cm to about4000 g-force/square cm.
 17. The shaped, bone fiber-based product ofclaim 1, wherein the shaped product is not a putty.
 18. The shaped, bonefiber-based product of claim 1, wherein a final shape of the product iswithin about 10% of a projected size of the product based on a mold tomake the product.
 19. The shaped, bone fiber-based product of claim 4,wherein the bone fibers comprise at least one growth factor of a IGF-1,a BMP-2, a BMP-4 or a BMP-7.
 20. A three-dimensional shape fiber-basedproduct of specific dimensions, comprising: a plurality of fibers,wherein an average length of the plurality of fibers is between about 1mm to about 200 mm and wherein a material for the plurality of fibers isselected from the group consisting of bone, wood, food stuffs,plant-based materials, elastomers, thermoplastics, and combinationsthereof.